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Showing: 361 - 380 of 902 results

  1. Deep Dive: Statistical Tests (Comparisons)

    Type
    Blog Post
    Published
    March 7, 2023, 2:15 p.m.
    ...to use them, what they can tell you, and how to identify which one is right for you. Statistics – The ...permutation of pairs within the five is different. To identify significant pairings, additional tests, referred...hoc test, done after your initial analysis can provide information on specifically which groups are statistically...within your population? Sometimes this can’t be avoided, but if it can be, you can reduce the family-wise... p value less than or equal to the alpha value divided by the number of null hypotheses. Benjamini uses...uses a complementary approach which identifies a p value cut off. This method entails arranging all calculated... by finding the largest p value where its rank divided by the total number of comparisons performed multiplied...

  2. CRISPR 101: Cytosine and Adenine Base Editors

    Type
    Blog Post
    Published
    Feb. 13, 2025, 2:15 p.m.
    ...DNA bases without breaking the DNA backbone, sidestepping the problem of HDR vs. NHEJ entirely. The first...hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com. How ...al., 2016). Target-AID acts similarly but not identically to BE3, modifying a 3-5 base window 18 bases ...editors with editing windows as small as 1-2 nucleotides. To reduce off-target effects associated with... created HF-BE3, a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3...positions 4-7. ABEs 6.3, 7.8, and 7.9 display slightly wider editing windows of position 4-9, although editing...Although the lab did not conduct comprehensive genome-wide studies of ABE specificity, their other experiments...

  3. Mouse Modeling, Part 1: Genetically Engineered Mice

    Type
    Blog Post
    Published
    July 11, 2019, 1:26 p.m.
    ... in genetically identical mice. Thus, scientists must genotype each animal to identify the ones with the...-lox Read our Genome Engineering Guide Learn more in our CRIPSR guide ...traits and genetically engineered mouse models provide researchers with useful and adaptable tools to ...background, which means that each mouse is genetically identical and all mice of the strain are homozygous at nearly...inserted gene may have errors introduced to the nucleotide sequence during the DNA repair process. The addition...reporter gene, which replaces the targeted gene and provides a way to track successfully engineered cells. ...

  4. Antibodies 101: Stripping and Reprobing Western Blots

    Type
    Blog Post
    Published
    Aug. 1, 2024, 1:15 p.m.
    ...You’ll want to set up your blot with a polyvinylidene difluoride (PVDF) membrane, which is sturdier and...Western blots are a great tool to identify a protein of interest in a complicated solution like cell ...already run your blot using nitrocellulose, you may decide you have nothing to lose by trying to strip and...abundance of your targets isn’t too different, consider antibody affinity: stronger binding antibodies...matter what stripping methods you throw at it. Ideally, dry your membrane once before any detection steps...(βME) 100–115 mM = 0.7%–0.8% (v/v) Breaks disulfide bonds HCl Adjust pH to 6.7–6.8 Adjust ...addgene.org Western Blot Protocol Western Blot Protocol Video ...

  5. Viral Vectors 101: Chemogenetics

    Type
    Blog Post
    Published
    July 11, 2023, 1:15 p.m.
    ...Chemogenetics has become a popular tool in the field as it provides a noninvasive method for manipulating cell activity... fast-acting optogenetic tools. The advantages provided by chemogenetics are instead that it is noninvasive... crossing the blood-brain barrier.  Another consideration when picking ligands for chemogenetic studies...response you aim to manipulate. It is important to consider that the same receptor can elicit different responses...muscarinic receptor derived DREADDs has been clozapine-N-oxide (CNO). However, concerns have been raised over the...Pharmacokinetic and pharmacodynamic actions of clozapine-N-oxide, clozapine, and compound 21 in DREADD-based chemogenetics... Gomez J, Ramirez M, Hu X, Galvan A, Basu J, Michaelides M, Sternson S (2019) Ultrapotent chemogenetics...

  6. Antibodies 101: Choosing the Right Isotype

    Type
    Blog Post
    Published
    April 6, 2023, 3 p.m.
    ...isotype choice. Target and application should be considered The first question you may have is whether a ...monoclonal primary antibody (A). To boost signal, consider using subclass specific secondary antibodies instead...strength (Fig. 1). Monoclonal primaries If you do decide on a monoclonal antibody you may wonder, “Will ...immunoprecipitations. Your sample species should also be considered when choosing an antibody. In some applications...options are available to you, it may be worth considering an antibody raised in a different species.    ...      Table 1: Isotype considerations when choosing antibodies   Complexity is a key factor...with a distinctly conjugated secondary antibody. Consider too that some secondary antibodies can cross react...

  7. Simple CRISPR-based Epigenetic Editing: dCas9-directed DNA Demethylation

    Type
    Blog Post
    Published
    Dec. 14, 2021, 2:15 p.m.
    ... 2021,  with over 70,000 articles in PubMed, the idea that DNA methylation is linked to disease has now...tools to specifically edit methylation sites.  Step aside, transcriptionally active dioxygenase  When I started...Recently, however, a particular tool has gained widespread popularity – and notoriety – as a tool of choice...   Let me leave you with a few practical considerations: Design your gRNA to overlap the CpG of interest...interest. Having the CpG directly inside the gRNA sequence is a great guarantee that DNMT1 will be blocked...Cas9 Resources at Addgene.org Read our CRISPR Guide to Epigenetics References Charlton J, Jung EJ, ...Galonska C, Charlton J, Mattei AL et al (2018). Genome-wide tracking of dCas9-methyltransferase footprints. ...

  8. Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene

    Type
    Blog Post
    Published
    April 6, 2021, 12:30 p.m.
    ...sequence or a sequence provided by the depositor. Depositing labs often provide insert sequences or annotated...to check the quality of other services that we provide, including viral preps and pooled libraries. As...this changeover had no impact on our ability to provide requesting scientists high quality data! Addgene's...culture to prepare it for sequencing, we feel confident that these few minor mismatches usually don’t ...differs from what we would expect given the data provided by the depositing lab, we call these “quality ...Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations...

  9. Reaching out to China: Canton Nucleic Acids Forum (CNAF) 2015

    Type
    Blog Post
    Published
    Dec. 1, 2015, 3:30 p.m.
    ..., loci were identified for 61 such diseases. Now single gene alleles have been identified for 4,163, over...surveillance of repetitive gene families and provide a way to guide inheritance that might be less error prone...plasmids, especially those of the CRISPR variety (see slides from my talk below). Largely organized by Dmitry...of items, only a small number of which I could identify. There were dozens of different mushroom varieties...how the NIH sees the future for genomics, and President Obama’s Precision Medicine Initiative and how ...shRNA silencing of one circular RNA they have identified resulted in a cell migration defect. They have...

  10. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    Published
    Sept. 24, 2024, 1:15 p.m.
    ...magnetoresponsive proteins. Motivated by curiosity Although a wide range of fluorescent proteins and optogenetic tools...possible only in non-biological conditions. So, they decided to search for a magnetic response among other proteins...Hayward et al. 2024 under CC-BY license.   Video 1: Fluorescence movie of E. coli expressing MagLOV...imaged area measures approximately 82 mm across. Video courtesy of Andrew York and Maria Ingaramo.   MagLOV...fluorescent proteins.  Currently, the team is working to identify how MagLOV’s structure changes under magnetic ...microscopy tools for biological applications across a wide range of topics. He embraces open science, self-... for?”, and while Andrew and Maria have lots of ideas and are happy to speculate, they know they can’t...

  11. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    Published
    Oct. 18, 2018, 12:37 p.m.
    ...with low fidelity polymerases and the cost of synthesizing longer oligos, which could be avoided using restriction...into E. coli for propagation. However, while not widely known, plasmid assembly can be achieved in vivo... of a plasmid, order primers that bind at either side of the undesired portion, designed to amplify the... Mutagenesis is performed by designing primers astride the old codon, amplifying the whole vector. The...homology, so performing more complex procedures can be aided by extending homologous sequences (to 25-30 bp)....colonies for simple cloning procedures. Use high fidelity DNA polymerase This will minimize random errors... blog posts Read our blog post on 5 factors to consider before choosing a cloning method Check out all...

  12. Lentiviral Vector Uses and Overview

    Type
    Blog Post
    Published
    May 19, 2016, 2:30 p.m.
    ...envelope have high infectivity in a wide range of cell types, so they’re ideal for work with primary cells or...envelope protein (Env), usually from VSV-G due to wide infectivity, is often used as a substitute for HIV..., used both for targeted modification and genome-wide screening.   Lentiviral vectors in the clinic Since... check out our lentiviral plasmid and biosafety guides before beginning your experiment. Let us know how...PMCID: PMC4273606. 4. Improved vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem...Genome-Scale, Knockout Screening Read about Genome-Wide Screening Using CRISPR/Cas9 Additional Resources...Resources on Addgene.org Check out Our Lentivirus Guide Learn about Virus Biosafety Find Lentiviral Vectors...

  13. Plasmid Preps: Different Purity, Different Quantities, Different Uses

    Type
    Blog Post
    Published
    June 16, 2020, 1:25 p.m.
    ...plasmid from a bacterial culture, it is important to consider your experiment.  It will dictate the amount of...260 and 230 nm ratios should fall ~2 - 2.2, and provide an indication of purity versus chaotropic agents...agents as guanidine thiocyanate and guanidine hydrochloride used during plasmid extraction. Figure...be entirely endotoxin free. Endotoxin is a lipopolysaccharide of the cell wall of Gram negative bacteria... For these reasons commercially available kits provide extra washing steps to favor the removal of this...want to utilize your plasmid DNA, so that you can decide what will be the optimal type of plasmid preparation...Brush up on molecular biology protocols using our video collection ...

  14. Neuronal labeling with Spaghetti Monster

    Type
    Blog Post
    Published
    Aug. 14, 2018, 12:42 p.m.
    ...recombinant Adeno-associated virus (AAV) has been widely used to deliver genetic tools to the brain because...neuronal circuits. Epitope tags are short antigenic peptide sequences, attached to a protein of interest (POI...simian virus 5-derived epitope (V5), the synthetic peptide FLAG, the synthetic streptavidin-binding strep-...both (Viswanathan, 2015). For Looger’s team, an ideal probe should combine the solubility, cell tolerance...imaging experiments, the second color is usually provided by red fluorescent proteins such as tdTomato or...smFPs can label fine neuronal structures with high fidelity and at lower concentrations than GFP. smFPs enable...N-cadherin in neurons and show that smFPs fusions provided better labels than fusions to 3 or more HA tags...

  15. Antibodies 101: The Four ELISAs and When to Use Them

    Type
    Blog Post
    Published
    July 25, 2024, 1:15 p.m.
    ...advantages and disadvantages of each, and some considerations when using the method. ELISA basics All ELISAs...methods. For enzymatic signal detection, users provide a substrate that the enzyme converts into a detectable...those studying low abundance antigens may want to consider alternative detection methods.      Figure...conjugated primary antibody. The reporter reacts with a provided substrate and produces a signal that is directly...can be challenging since scientists must first identify two independent antibodies that work well together... of conjugated antibodies   Additional considerations No matter what ELISA method you choose, it ...sample of wild type cells, purified protein, or a peptide. The positive control will confirm that the procedure...

  16. Antibodies 101: Immunoprecipitation

    Type
    Blog Post
    Published
    Dec. 7, 2021, 2:15 p.m.
    ...When choosing beads, there are several factors to consider. Agarose beads are sponge-like and vary in shape...shape and structure. Their porous surface provides a large area for binding, but antibodies conjugated ...to the capture antibody and is immobilized. The ideal antibody concentration to use will vary between ...antibody is functioning as expected. In order to identify any non-specific interactions, perform parallel... is used to isolate the protein of interest. The wide availability and high specificity of anti-epitope...appealing, but the disadvantages must be carefully considered before using. For example, the tag may affect...routinely use the chromatin IP (ChIP) technique to identify DNA binding proteins involved in histone modification...

  17. Career Insights: Technical Support Specialist

    Type
    Blog Post
    Published
    Nov. 9, 2017, 2 p.m.
    ... live it out, but ideally without asking too many (annoying) questions. Identifying and solving problems...was aware that such roles existed, but did not consider them for myself. I started picturing myself in...Other Addgenies and Their Careers in Our Career Videos What's required for a role in technical support...any incoming requests or problems - there is considerable diversity in the types of questions customers...consuming and/or less technically challenging. Alongside there is a second-line team that takes on cases...problem. It may not always be straightforward to identify those crucial details because there are just so...Visit the Addgene Careers Page Check Out Our Career Videos ...

  18. How to Be an Excellent Trainee

    Type
    Blog Post
    Published
    Sept. 26, 2023, 1:15 p.m.
    ...though. Typically, you will identify a ‘scientific mentor’: someone who provides career advice and offers... and identify any gaps or questions you have, then bring them to your mentor. Pro tip! Consider physically... a quality mentor, and rightfully so! They can provide you with high-level career advice, open doors for...Training follow up After your training session (ideally in the next few days or weeks), look over your ...protocol, or maybe you would like to take pictures/videos of a machine set up while your mentor is demonstrating..., your bench mentor’s training time is time set aside just for you – so do your best to show up as your...

  19. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    Published
    June 28, 2016, 10:59 a.m.
    ... will outline a simple method for selecting the guide RNA, validating its efficacy in vitro, and using...d like to manipulate in mind. Now it’s time to consider where you would like to attack this gene. Should... When the target sequence was digested by sgRNA guided Cas9 endonuclease, homology dependent repair (HR... It can take roughly 3 months from the day you decide to do a gene modification experiment in mice to...have your F0 generation (barring life’s little accidents and stuff ups). This is a huge improvement on ...production of mouse lines that could be used to study a wide variety of phenomena, it’s possible that we’ll learn...of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases." Genome research 24.1...

  20. Getting the Most from Your Lentiviral Transduction

    Type
    Blog Post
    Published
    April 7, 2016, 2:30 p.m.
    ... in lower lentiviral titers. Another factor to consider is the age of your culture post thaw. In general...lower titer virus. In such cases you may want to consider concentrating your viral prep. Storage can also.... For long-term storage, viral preps should be divided into single-use aliquots, and stored at -80°C. ... start to a successful infection, one point to consider is how the virus is being titered. Most titering... that routinely produce lentivirus may want to consider testing a variety of cation and serum sources .... “Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention...About Viral Vector Elements Learn How to Do Genome-Wide Screens with CRISPR Lentiviral Vectors Additional...
Showing: 361 - 380 of 902 results